Human immunodeficiency virus (HIV) infects an estimated 34 million individuals worldwide, leading to the deaths of over 30 million since its identification and nearly 2 million in 2011 alone. HIV has been immensely difficult to combat largely because of its propensity to quickly mutate, permitting rapid evolution of drug resistance and immune escape. Furthermore, multiple cross-species transmission events and founder effects have led to the existence of distinct types, groups, and subtypes of HIV, further complicating treatment and hindering vaccine development. However, the impact of HIV type, group, and subtype on even the most basic of virological properties still often remains unclear. We propose here to examine the impact of HIV type, group, and subtype on viral mutagenesis, a key driver of viral evolution. Additionally, we propose to explore the ability of opioids to alte viral mutagenesis, as many individuals are simultaneously exposed to HIV and opioids, either through drug abuse or the legitimate use of opioids for managing pain. The impact of these factors on viral mutagenesis will be analyzed using Illumina sequencing of viruses subjected to a single round of replication, which will allow for a detailed and systematic comparison of mutation rates, spectra, and hotspots. Overall, the proposed research will determine the extent to which type/group/subtype and opioid use alter viral mutagenesis, as well as providing a powerful re-examination of much earlier studies on the mutagenesis of HIV. Ultimately, we anticipate that our research will shed light on factors that influence viral mutagenesis and aid in the customization of antiviral therapy for HIV-infected individuals based upon their infecting subtype(s) as well as whether they abuse opioids. Furthermore, the proposed research may have implications on the legitimate use of opioids for pain management in HIV-infected individuals.